Monoclonal antibodies to transforming growth factor-beta and methods of use

ABSTRACT

Monoclonal antibodies to transforming growth factor beta (TGF-B) are prepared from hybrid cell lines by immunizing with TGF-B2. The antibodies may be of any isotype and may be of any mammalian origin such as murine or human origin. Therapeutic applications for treating or reducing the likelihood of developing acute or chronic fibrosis utilizing the TGF-B monoclonal antibody are also disclosed.

This application is a division of application Ser. No. 07/759,109, filedSep. 6, 1991, now U.S. Pat. No. 5,571,714, which is a continuation ofapplication Ser. No. 07/288,432, filed Dec. 22, 1988, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of immunology andparticularly to monoclonal antibodies specific to transforming growthfactor beta (TGF-B) and applications thereof.

BACKGROUND OF THE INVENTION

Transforming growth factor beta (TGF-B) is a family of relatedmultifunctional regulators of cell growth which can affect cellularproliferation and differentiation. Two forms of TGF-B, TGF-B1 andTGF-B2, are, in general, multifunctional cytokines that have potentinhibitory effects on the proliferation and effector responses ofmitogen-, lymphokine-, and alloantigen-activated lymphocytes (Kearl etal, J Exp Med (1986) 163:1037; Ellingsworth et al, Cell Immunol (1988)113: in press; Kearl et al, J Immunol (1986) 137:3855; Ranges et al, JExp Med (1987) 166:99l). In addition, TGF-B1 and TGF-B2 affect theproliferation and differentiation of other cells of the immune systemincluding macrophages (Wall et al, Proc Natl Acad Sci USA (1987)84:5788), pre-B cells (Lee et al, J Exp Med (1987) 166:1290),hematopoietic stem cells (Ohta et al, Nature (1987) 329:539; Ishibashiet al, Blood (1987) 69:1737; Keller et al, J Exp Med (1988) in press;Sing et al, Blood (1988) in press), and NK cells (Rook et al, J Immunol(1986) 136:3916).

The generation of immunoprecipitating and neutralizing antibodies tonative TGF-B has been extremely difficult due to the highly conservednature of native TGF-B among different species. The human sequence ofTGF-B1 is identical to the bovine and porcine sequences and differs fromthe murine sequence by one amino acid. TGF-B1 is a dimer composed of twoidentical disulfide-linked chains of 112 amino acid residues.

TGF-B2 is also a dimeric polypeptide and is disclosed in U.S. Pat. No.4,774,322, filed Dec. 10, 1987, assigned to Collagen Corporation. Eventhough there are 14 amino acid differences in the first 36 amino acidsresidues of the two forms, their biological activities are similar(Cheifetz et al, Cell (1987) 48:409-415; Seyedin et al, J Biol Chem(1987) 262:1946-1949).

Western blots and immunohistochemical localization studies on TGF-BLhave been performed using a polyclonal rabbit antiserum obtained byimmunization with a synthetic peptide corresponding to the NH₂ -terminal30 amino acids of TGF-B1 (Ellingsworth et al, J Biol Chem (1986)261:12362). Polyclonal antisera to human and porcine TGF-B (Keski-Oja etal, Cancer Res (1987) 47:6451-6458) and to porcine TGF-B2 (Rosa et al,Science (1988) 239:783-785) have been shown to neutralize the biologicalactivity of TGF-B1 and TGF-B2, respectively. Monoclonal antibodiesspecific to TGF-B have not been previously described. The availabilityof these specific antibodies is important for characterization of therole played by TGF-B in the immune system and other physiologicalprocesses.

Antibodies to TGF-B are needed to investigate the varied biologicalactions of TGF-B, to study TGF-B biosynthesis, and to determinedifferences in activity or effect, if any, between the forms of TGF-B.These antibodies would also have therapeutic applications for treatingindications where there is an overproduction of TGF-B (e.g., acute liverinjury, chronic hepatic fibrosis) and for diagnosing or treatingmalignancies (e.g., sarcomas and melanomas) and metastatic cancers.

SUMMARY OF THE INVENTION

The present invention provides a monoclonal antibody immunoreactive withTGF-B. A monoclonal antibody is described herein that is specific forTGF-B. This antibody, in preferred embodiments, binds to a neutralizingepitope on TGF-B or binds to an epitope on TGF-B which blocks thebinding of TGF-B to its cellular receptors.

Another aspect of the invention is to provide a stable cell line whichsecretes a monoclonal antibody specific for TGF-B. In one embodiment ofthe invention, the cell line secretes a murine monoclonal antibodyspecific for TGF-B that has TGF-B neutralizing activity.

Another aspect of the invention is a method for treating acute andchronic disease states that result from an overproduction of TGF-B, byadministering a therapeutically effective amount of a monoclonalantibody reactive with TGF-B, or an antigen-binding fragment of amonoclonal antibody reactive with TGF-B.

Another aspect of the invention is a method for treating tumor cellsthat produce TGF-B, by administering a therapeutically effective amountof a monoclonal antibody reactive with TGF-B to suppress theimmunosuppressive effects of TGF-B.

Another aspect of the invention is a method for treating metastaticcancers by administering a therapeutically effective amount of amonoclonal antibody reactive with TGF-B to mark tumor cells fordestruction by complement or by immune cells dedicated to tumor cellremoval.

Another aspect of the invention is a capture-ELISA (CELISA) fordiagnosing disease states such as cancer and connective tissue(fibrotic) disorders comprising: coating a first monoclonal orpolyclonal antibody reactive with TGF-B onto a surface, adding a samplecontaining an unknown amount of TGF-B or a standard concentration ofpurified TGF-B to the surface, adding polyclonal sera reactive withTGF-B or a monoclonal antibody of the invention to the surface, addingan enzyme-linked tertiary antibody (reactive to said monoclonal orpolyclonal antibody), and determining the presence of TGF-B byquantifying the amount of enzymatic label present in a colorimetricreaction.

Another aspect of the invention is a chimeric monoclonal antibody,having an antigen-binding portion derived from the MAb of the invention,and the remainder derived from human antibodies. Such chimericantibodies are described by S. L. Morrison, Science (1985) 229:1202.

Another aspect of the invention is an anti-idiotypic antibody reactivewith the MAbs of the invention, which are useful as TGF-B mimics. Thesemimics are capable of binding to TGF-B receptors, and thus may act asTGF-B agonists or antagonists.

These and other objects and features of the invention will become morefully apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a Western blot of monoclonal antibodies bodies binding topurified TGF-B2. Lanes 1-6 and 7-12 contain TGF-B2 run undernon-reducing and reducing conditions, respectively.

FIG. 2 illustrates the neutralization of TGF-B2 activity by the lD11.16MAb.

FIG. 3 represents an autoradiogram showing inhibition of affinitycross-linked cell receptors from both thymocytes and the NRK cell lineby MAbs.

FIG. 4 illustrates the immunoprecipitation of ¹²⁵ I!-labeled TGF-B2.

FIG. 5 is a Western blot of monoclonal antibody 1D11.16 binding topurified TGF-B1 and TGF-B2. Lanes 2-5 and 6-9 contain various amounts ofTGF-B2 and TGF-B1, respectively, run under non-reducing conditions.

MODES OF CARRYING OUT THE INVENTION

The present invention is directed broadly to the production of TGF-Bmonoclonal antibodies, neutralizing TGF-B antibodies and TGF-Bantibodies that block the binding of TGF-B to cellular receptors. Thepresent invention is also directed to the use of such antibodies indiagnostic assays to determine elevated levels of TGF-B that areassociated with certain disease states, and in therapeutic applicationsto prevent the overproduction of TGF-B or to suppress theimmunosuppressive effects of TGF-B in tumor cells.

A. Definitions

As used herein the terms "monoclonal antibody" and "MAb" refer to animmunoglobulin composition having a substantially homogenous populationof antibodies, each of which binds to the same antigenic determinant.Unless otherwise indicated, the term is not intended. to be limited toantibodies of any particular mammalian species or isotype, or toantibodies prepared in any given manner. The term is intended to includewhole antibody molecules as well as antigen-binding fragments (e.g.,Fab', F(ab')₂).

"Cell line" refers to various embodiments including but not limited toindividual cells, harvested cells, and cultures containing cells so longas they are derived from cells of the cell line referred to.

As used herein with respect to characterizing the claimed hybrid celllines, the terms "permanent" and "stable" mean that the lines remainviable over a period of time, typically at least about six months, andmaintain the ability to produce the specified monoclonal antibodythrough at least about 50 passages.

"TGF-B" refers in general to the family of transforming growth factors(beta-chain) which participate in the control of cell proliferation anddifferentiation. As used herein the term refers to both TGF-B1 andTGF-B2 proteins. Other known TGF-B-like proteins are the "inhibins"(Mason et al, Nature (1985) 318:659-663), their beta-chain dimers,"activins" (Ling et al, Nature (1986) 321:779-782; Vale et al, Nature(1986) 321:776-779), Mullerian inhibiting substance (Cate et al, Cell(1986) 45:685-698) and a predicted protein product of thedecapentaplegic gene complex of Drosophila (Padgett et al, Nature (1987)325:81-84) as shown by complementary DNA sequence analysis.

An antibody that "is reactive with" or "specific to" TGF-B is anantibody that binds an epitope present on TGF-B.

An antibody that "neutralizes in vitro biological activity of TGF-B"when bound to its epitope on TGF-B is defined by testing theantibody-bound TGF-B in an in vitro assay such as the IL-1 andPHA-dependent thymocyte proliferation assay, the NRK proliferation assayor an assay using the ROS 17.2.8 osteosarcoma cell line, all of whichare described in the following examples.

An antibody that "blocks binding to cellular receptors" of TGF-B is anantibody that blocks the binding of TGF-B to TGF-B cellular receptors ina standard receptor binding assay, such as the one described in theexamples below. Cells which are known to have TGF-B cellular receptorsinclude mouse thymocytes (C3H/HeJ) and the NRK cell line (clone 49F,ATCC CRL 1570).

The term "binding affinity" or "K_(a) " of an antibody to its epitope,as used herein, refers to a binding affinity that may be calculatedaccording to standard methods by the formula K_(a) =8/3 (It-Tt) where Itis the total molar concentration of inhibitor uptake at 50% tracer andTt is the total molar concentration of tracer. See Muller, J ImmunolMeth (1980) 34:345-352. Binding affinity may also be calculated usingthe formula B/T=n·N_(Ab) ·W· (V--V_(m))K+Q·W!. See Antoni and Mariani, JImmunol Meth (1985) 83:61.

The term "therapeutically effective amount" as used herein refers to theamount of antibody that neutralizes the biologic activity of TGF-B,which may be measured by either (1) a pathologic evaluation of theprevention of fibrosis, or (2) an inhibition of the immunosuppressiveeffects of TGF-B which results in tumor regression.

The general procedures of producing monoclonal antibodies, including thecell lines which produce such compositions, are well known in the art.See, e.g., Gerhard et al, Proc Natl Acad Sci USA (1978) 75:1510;Monoclonal Antibodies (R. Kennett, T. McKearn, & K. Bechtol eds. 1980);Monoclonal Antibodies and T-Cell Hybridomas (G. Hammerling, U-Hammerling, & J. Kearney eds. 1981); Kozbor et al, Proc Natl Acad SciUSA (1982) 79:6651; Jonak et al, Hybridoma (1983) 2:124; MonoclonalAntibodies and Functional Cell Lines (R. Kennett, K. Bechtol, & T.McKearn eds. 1983); Kozbor et al, Immunology Today (1983) 4:72-79;Shulman et al, Nature (1982) 276:269-270; Oi et al, in Selected Methodsin Cellular Immunology, pp. 351-371 (B. Mishell & S. Schiigi eds 1980);and Foung et al, Proc Natl Acad Sci USA (1983) 79:7484-7488.

In a preferred embodiment, anti-TGF-B antibody-producing primary B cellsare isolated from a mammal immunized with TGF-B and then immortalized sothat a cell line can be established. Any appropriate technique ofimmortalizing the primary B cells can be employed, including, but notlimited to, fusion with a myeloma cell, transformation with oncogenicDNA, or transfection with Epstein-Barr virus. Preferred myeloma cellsfor this purpose are those which do not themselves secreteimmuno-globulins, which fuse efficiently, support stable high-levelexpression of antibody by the selected antibody-producing cells, and aresensitive to selection mediums such as RAT medium. Among these,presently preferred myeloma cell lines are murine myeloma lines such asthose derived from P3X63-Ag8.653 (653) and Sp2/0-Agl4 (SP2/0) myelomalines available from the American Type Culture Collection, Rockville,Md. under ATCC CRL Nos. 1580 and 1581, respectively.

Immortalized cells, such as the murine hybridomas, are screened forproduction of the desired anti-TGF-B antibodies having the appropriateaffinity and/or epitope. A particularly useful screen for regularlyisolating high affinity antibodies is to screen antibodies using a solidphase radioimmune assay (RIA). Generally, monoclonal antibodies havingbinding affinities for TGF-B of at least about 10⁷ M⁻¹ are selected. Thepresent invention regularly permits the isolation of antibodies withbinding affinities of at least about 10⁷ M⁻¹ to about 10⁸ M⁻¹.

The present invention is also directed to anti-TGF-B monoclonalantibodies that neutralize the in vitro inhibitory effects of TGF-B.Screening monoclonal antibodies for this ability can be in addition tothe screen for high binding affinity. In general, standard in vitroneutralization assays may be employed in this screen. In a preferredembodiment of the present invention, the MAb 1D11.16 is found toneutralize both TGF-B1 and B2 activity. This suggests that a conservedportion of both molecules serves as the MAb binding epitope, and thatthis epitope is either within or close to the cell receptor binding siteof TGF-B. Work by Flanders et al, Biochem (1988) 27:739-746 withpolyclonal antisera raised against synthetic peptides corresponding todifferent regions of TGF-B1 implicated the C-terminal portion of TGF-B1in receptor binding. Comparison of the C-terminal sequences of TGF-B1and B2 reveals only 7 differences in the final 30 amino acid residues ofthe molecules. Since the most highly conserved portion of the moleculesis actually between residues 20 and 44, it appears possible that thisregion is a target for the cell receptor binding site on TGF-B.

Several preferred murine monoclonal antibodies have been producedaccording to the present invention. Monoclonal antibody 1D11.16 (IgGl)refers to the MAb produced by a clone of the murine hybridoma cell line1D11.16. Samples of this cell line were deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. Cellline 1D11.16 was assigned ATCC HB9849, and is guaranteed to be publiclyavailable for thirty years after the issue date of this patent.Monoclonal antibodies produced by the clones of this cell line may bedistinguished using the Western Blot Assay, wherein the antibody MAb1D11.16 would recognize both TGF-B1 and TGF-B2. The antibodies of thepresent invention may be used to treat acute or chronic fibrosis that isassociated with overproduction of TGF-B. The body responds to injury ordisease by regenerating destroyed tissue. When the injury or disease isprolonged or extensive, the destroyed tissue may be replaced byspecialized fibrotic connective tissue. Excess deposition of thisfibrotic tissue may result in an impairment of the affected tissue ororgan function, and in some instances may be disfiguring to the patient.It is currently believed that there is an overproduction of TGF-B indiseases such as interstitial lung fibrosis, liver cirrhosis, andfibrotic skin disorders such as scleroderma and scarring: M. J. Czaja etal, J Cell Biol (1988) (in press); D. G. Hoyt et al, J Pharm Exp Ther(1988) 246:765. A therapeutically effective amount of the antibody ofthis invention may be administered to neutralize the biologic activityof TGF-B, which in turn would result in prevention of unwanted fibrosis.

It is also generally known that TGF-B is produced by different tumorcells (sarcomas and carcinomas). This production may protect the tumorcells from recognition by the host's immune system: M. Wrann et al, EMBOJ (1987) 6:1633. In these situations, TGF-B suppresses the proliferationof T and B cells, NK cells, LAK cells, and macrophages that are involvedin tumor destruction. The MAb of this invention may be administered toblock TGF-B's immunosuppressive effects (to permit the generation of animmune response against the tumor), and result in tumor regression.

The antibody of the invention neutralizes the biological activity ofTGF-B by preventing the antigen from binding to its cell surfacereceptors. The intact antibody, antigen binding fragments (e.g., Fab',F(ab')₂), or chimeric antibodies may be useful in these applications. Inaddition, administration of the antibodies of the invention would formimmune complexes (antigen-antibody complexes) that will increase therate at which the antigen TGF-B, is cleared from systemic circulation orfrom the tissue site where the antigen is produced.

These types of fibrotic diseases and tumor cells may be treated byadministering a therapeutically effective amount of the antibodies ofthe invention to affect the inhibition of fibrosis formation orregression of tumor cells. The method and frequency of administration,the dose range, and the duration of the antibody therapy will vary withthe severity and nature of the condition, and the general health of thepatient.

In a preferred embodiment, the antibodies of the invention areadministered locally to the affected tissue sites by bolus injection orperfusion. The amount of antibody administered may be measured bymaintaining the local tissue concentration of TGF-B at about 1-1,000 ug/mL.

Indications where this mode of treatment is particularly useful are forthe control of excessive scar tissue formation, due to surgery ortrauma, or prevention of the formation of connective tissue adhesions.For treatment of tumor cells by local administration, the antibodies maybe delivered directly into a tumor mass through a vascular catheter fordeep solid tumors, or through a hypodermic needle for superficial orcutaneous tumors. The antibodies may be locally administered by a singlebolus injection that is repeated over several days, or by continuousadministration by perfusion. The amount of antibody administered ispreferably about 1 ug to 1,000 ug per gram tumor tissue.

In another preferred embodiment, the antibodies of the invention may beadministered systemically by intravenous or peritoneal perfusion, or bybolus injection into the subcutaneous tissue or muscle. The antibodiesmay be delivered in vehicles generally known to those skilled in theart, such as saline, balanced salt solutions, isotonic or phosphatebuffered saline (pH 7), with or without dextrose. The amount of antibodyadministered may be measured by maintaining the circulating serumconcentration between about 1 ug to 5 mg per mL serum.

Indications where this mode of treatment is particularly useful aresystemic diseases such as interstitial lung fibrosis, liver cirrhosis,scleroderma, and metastatic cancer.

For both local and systemic treatment, the antibodies of the inventionmay be administered in combination with other antibodies reactive withTGF-B to reduce the amount of bioavailable factor.

The antibodies of the present invention may also be used in a number ofimmunoassays, including assays to purify or quantify TGF-B. For example,in one embodiment, a monoclonal antibody reactive with TGF-B, such as1D11.16, is coated onto microtiter wells. This antibody will capture anyTGF-B present in a solution of unknown proteins. The captured TGF-B isthen recognized by a second polyclonal antibody that also recognizesTGF-B. The second antibody is preferably of a different subclass orspecies than the first antibody, to allow the specific recognition ofthe second antibody by a third antibody (e.g., goat anti-rabbit IgG)that is coupled to peroxidase. Thus, in this assay, color developmentonly occurs when TGF-B binds to the capturing antibody 1D11.16. Theamount of TGF-B in an unknown solution is quantified by comparison ofcolor development to a standard dilution curve of known amounts ofTGF-B. High levels of TGF-B are indicative of the presence of fibroticdisease or tumor mass.

The following examples are presented for illustrative purposes only andare not to limit the scope of the present invention variations in thesource, type, and method of producing antibodies will be apparent tothose of ordinary skill in the art, and may be employed withoutdeparting from the scope of the present invention.

EXAMPLE 1 (Purification of bovine TGF-Bs)

Bone derived TGF-BL and B2 were purified from the noncoliagenous,guanidine-HCl soluble protein fractions of demineralized bovinemetatarsal bone. These factors were purified by a combination of gelfiltration, CM-cellulose cation exchange chromatography and C18:reversephase high pressure liquid chromatography (HPLC) using a previouslydescribed method (Seyedin et al, Proc Natl Acad Sci USA (1985) 82:2267).Protein concentrations were determined spectrophotometrically bycomparison of absorbance at OD₂₁₀ to a solution of protein of knownconcentration.

EXAMPLE 2 (Immunization of mice and fusion procedure)

A group of female Balb/c mice was injected intraperitoneally (IP) with10 ug of native bovine TGF-B2 with complete Freund's adjuvant (SigmaChemical Co., St. Louis, Mo.). At 3 week intervals, the animals wereboosted with 10 ug of TGF-B2 in incomplete Freund's adjuvant (Sigma).Following the second boost, the mice were bled and serum antibody titersagainst TGF-B2 checked by ELISA. The animal with the highest titer(recip. dil.=5120) was given a final intravenous (IV) injection ofTGF-B2 three weeks after the last IP boost and sacrificed for use in thefusion process. For all injections, the TGF-B2 was lyophilized withcarrier mouse serum albumin (10 ug albumin per ug TGF-B2) prior tosolubilization and injection.

Fusion to the SP2/0 myeloma (GM3659 B, NIGMS Human Genetic Mutant CellRepository, Camden, N.J.) was performed 4 days after the IV challenge.The fusion was performed essentially according to the protocol of Oi andHerzenberg, "Immunoglobulin-producing hybrid cell lines" in SelectedMethods in Cellular Immunology, Mishell and Shiigi, eds., W. H. Freemanand Co., San Francisco, pp. 357-362, (1980). Spleen cells were mixedwith SP2/0 at a ratio of 5:1 (2.2-10⁸ splenocytes: 4.4-10⁷ SP2/0 cells).50% polyethylene glycol 1500 (Boehringer-Mannheim Biochemicals,Indianapolis, Ind.) was used as the fusagen. Cells were plated at 10⁶cells/well along with resident peritoneal cells at 4×10³ cells/well inDMEM with high glucose (4.5 g/l) supplemented with 20% FCS (HycloneLaboratories, Logan, Utah), 2 mM L-glutamine, 2 mM sodium pyruvate,nonessential amino acids, penicillin and streptomycin. In thisprocedure, aminopterin was replaced by azaserine (Sigma) according tothe procedure by Larrick et al, Proc Natl Acad Sci USA (1983) 80:6376,and added along with thymidine and hypoxanthine on day 1 after thefusion. In this fusion, virtually every well containedselection-resistant colonies that were screened for anti-TGF-B2 MAbproduction by ELISA. All wells positive for TGF-B2 MAb were cloned bylimiting dilution.

EXAMPLE 3 (ELISA assay)

The ELISA for TGF-B2 was performed according to standard procedures(Ellingsworth et al, J Biol Chem (1986) 261:12362; Engvall and Perlmann,J Immunol (1972) 109:129). Briefly, wells of a 96 well microtiter plate(Immulon, Dynatech Laboratories, Alexandria, Va.) were coated overnightwith 0.25 ug/well of TGF-B2 in a 0.01M carbonate coating buffer (pH9.6). For ELISAs done with TGF-B2 peptides, 2.5 ug/well of peptide wascoated onto plates overnight. Plates were washed with PBS (pH 7.4)containing 0.05% Tween® 20 (J. T. Baker, Phillipsburg, NJ) (PT buffer).Plates were blocked with PT containing 1% (w/v) gelatin for 60 min atroom temperature. After addition of culture supernatants or purifiedantibody for 60 min, plates were washed and incubated with 2nd stepantibody (peroxidase-conjugated goat anti-mouse IgG, SouthernBiotechnology, Birmingham, Ala.). After an additional 60 min, plateswere washed and incubated with a one component peroxidase substratesolution (ABTS) (Kirkegaard and Perry, Gaithersburg, Md.). Plates wereread with a titretek Multiscan MCC ELISA reader (Flow Laboratories,McLean, Va.). Identification of MAb subclasses was accomplished by ELISAas taught above, except that subclass specific secondary Abs wereemployed (rat anti-mouse MAb-peroxidase conjugates, Zymed, South SanFrancisco, Calif.).

Table I below summarizes some of the characteristics of each of theseMAbs- Of particular interest are the relative affinity measurements ofthe binding of the MAb to TGF-B2 as assessed by a solid phasecompetitive RIA. The MAb lDll.16 exhibits the highest affinity forTGF-B2. However, the affinities of all the MAb are relatively similar.Also of interest is the fact that only one of the MAbs, 1D11.16,cross-reacts with TGF-B1 by ELISA.

                  TABLE I                                                         ______________________________________                                        Properties of Anti-TGF-B Monoclonal Antibodies                                                          Neutral-                                                             X-reacts izing  Relative                                     MAb    Subclass  with B1  Activity                                                                             Affinity                                                                             Imppt.                                ______________________________________                                        3C7.14 2B        -        -      +++    ++++                                  1D11.16                                                                              1         +        +      ++++   ++                                    2G1.1.12                                                                             1         -        -      ++     +++                                   2D7.20 2A        -        -      +      +                                     ______________________________________                                         a Subclasses were determined by ELISA.                                        b Crossreactions were determined by ELISA with TGFB1 coated plates.           c Neutralization was determined in the Il1, PHAdependent thymocyte assay      (see FIG. 2).                                                                 d Affinity constants were determined by solid phase RIA and nonlinear         regression analysis. Four + indicates MAb with highest affinity. Ka           determined from three experiinents are as follows: 1D11.16, 3.4 ×       10.sup.8 L/mol; 3C7.14, 1.9 × 10.sup.8 L/mol; 2G1.1.12, 1.6 ×     10.sup.8 L/mol; and 2D7.20, 1.6 × 10.sup.7 L/mol.                       e Immunoprecipitation was performed by mixing  .sup.125 Ilabeled TGFB2        with MAb and followed by addition of antiIg-agarose (see FIG. 3). Four +      indicates MAb with highest precipitating capability.                     

Mapping of the epitopes recognized by the MAbs was attempted by runningELISAs with synthetic peptides corresponding to portions of TGF-B2 ascoating antigens. Three peptides were tested: 1-29, 47-76, 79-108. Noneof the MAbs were found to bind appreciably to the synthetic peptideseven though a 30-fold molar excess of peptide was coated relative to theamount of TGF-B2 normally coated onto plates. A possible explanation forthis result may be provided by the Western analysis (see below). In theWesterns, only the MAb 2G1.12 weakly recognized the reduced monomericform of the molecule.

EXAMPLE 4 (Western Blots)

This protocol follows standard procedures (Ellingsworth et al, (1986)supra; Towbin et al, Proc Natl Acad Sci USA (1979) 76:4350. TGF-B2 waselectrophoresed in a 15% SDS polyacrylamide gel. The protein was thentransferred to nitrocellulose in a high pH transfer buffer (25 mM Tris,192 mM glycine, pH 11; with 20% methanol). After overnight transfer in aBio-Rad transfer apparatus (170 mA constant current), the blot wasblocked with 2.5% non-fat dry milk in PT (PT-BLOTTO). Thenitro-cellulose strips were incubated with MAb-containing supernatantsor purified IgG in PT-BLOTTO for 60 min. After washing, the strips wereincubated with the previously-described 2nd step antibody(peroxidase-conjugated goat anti-mouse IgG) for an additional 60 min.The presence of antibody was visualized by addition of the peroxidasesubstrate: 0.06% (w/v) 4-chloro-1-napthol (Sigma) and 0.01% hydrogenperoxide in 0.05 M Tris-buffered saline (pH 7.4). The reaction procedurewas halted by extensive washing of the blot in deionized H₂ O.

FIG. 1 illustrates the Western blot pattern of the binding of the fourMAbs to native bovine TGF-B2. Lanes 1-6 and 7-12 contain TGF-B2 rununder non-reducing and reducing conditions, respectively. Lanes 1 and 7are amido black stained TGF-B2- Lanes 2-6 and 8-12 were incubatedrespectively with the following antibodies: lanes 2 and 8, 2F9-15; lanes3 and 9, 3C7.14; lanes 4 and 10, 1D11.16; lanes 5 and 11, 2D7.20; andlanes 6 and 12, 2G1.1.12. The monoclonal 3C7.14 is the strongest reactorwith TGF-B2 in these blots. Although not visible on the gel, MAb2G1.1.12 reacted very slightly with reduced monomeric TGF-B2; none ofthe other MAbs are able to bind appreciably to reduced monomeric TGF-B2(lanes 9-12). Also included in lanes 2 and 8 is the reactivity patternof an additional MAb that recognizes a protein contaminant present inthe biochemically purified TGF-B2 preparations. This pattern isrepresentative for several other MAbs produced that also recognize thisidentical contaminant (data not presented). On silver staining of PAGEgels, this contaminant was found to represent a small percentage of theimmunizing protein.

FIG. 5 illustrates the binding of MAb 1D11.16 to both TGF-B1 and TGF-B2under non-reducing conditions Lanes 2-5 and 6-9 contained variousamounts of TGF-B2 and TGF-B1, respectively. The amounts were 1 ug/lane(lanes 2 and 9), 0.25 ug/lane (lanes 3 and 8), 0.1 ug/lane (lanes 4 and7), and 0.025 ug/lane (lanes 5 and 6). The lack of reactivity in lane 5is believed to be an artifact due to loss of the sample duringlyophilization prior to running the SDS gel.

EXAMPLE 5 (Neutralization of bioactivity)

The IL-1 and PHA-dependent thymocyte assay for TGF-B described inEllingsworth et al, Cell Immunol (1988) 114:41 was employed. In theseexperiments, either 50 or 100 ul of MAb-containing culture supernatantor purified IgG diluted in medium was added to the cultures at the sametime as TGF-B1 or B2. The TGF-B concentrations were employed over arange of 10⁻¹² to 10⁻¹⁰ M.

Preliminary experiments showed that only one of the four MAbs, 1D1l.16,was able to neutralize the inhibitory effects of TGF-B. FIG. 2 shows theability of affinity purified 1D11.16 to neutralize theanti-proliferative actions of TGF-B. This figure demonstrates that theIL-1 and PRA-dependent proliferation of thymocytes is inhibited byTGF-B2 over a concentration range of 10⁻¹² to 10⁻¹⁰ M. Addition of 10 or100 ug/mL 1D11.16 will effectively neutralize 10⁻¹¹ M TGF-B2 when bothTGF-B2 and antibody are added at the start of culture. 1D11.16 is alsofound to neutralize the activity of TGF-B1 to a similar extent in thisassay. This suggests that a similar epitope on both TGF-B1 and B2 isrecognized by the neutralizing antibody and that this epitope may bewithin or adjacent to the receptor binding site on TGF-B. 1D11.16 isable to neutralize the growth promoting actions of TGF-B1 and B2 in theNRK proliferation assay (Roberts et al, "Purification of type Btransforming growth factors from non-neoplastic tissues" in Cell CultureMethods for Molecular and Cell Biology, Barnes et al, eds., Ed. AlanLiss, Inc., New York pp. 181-194, (1984)) and to neutralize TGF-Binduced alkaline phosphatase production in an assay using the ROS 17.2.8osteosarcoma cell line (obtained from Dr. Greg Mundy, U. Texas HealthScience Center, San Antonio, Tex.). These results suggest that a singlereceptor binding domain is present in TGF-B1 and B2 and that thisbinding domain appears to be responsible for the pleiotropic actionsattributed to TGF-B1 and B2.

EXAMPLE 6 (Inhibition of receptor cross-linking by 11.16)

The cross-linking of ¹²⁵ I)-labeled TGF-B1 and B2 to its cellularreceptors employs the methods described in Ellingsworth et al (1986)supra and Segarini et al, J Biol Chem (1987) 262:14655. In the currentexperiments, either 1D11.16 antibody (30 ug/mL) or a non-TGF-B-reactivemouse MAb at an equivalent concentration was pre-mixed with ¹²⁵I!-labeled TGF-B1 or B2 (50 pM final concentration) for 60 min prior toaddition to the cells. The cells employed in the assay were freshlyisolated mouse thymocytes (C3H/HeJ) and the NRK cell line (clone 49F,CRL 1570).

The ability of the MAb 1D11.16 to block the cross-linking of ¹²⁵I!-labeled TGF-B by addition of disuccinimidyl suberate (DSS) to itscell surface receptor complex was assessed. The receptor complexconsists of three major species that have approximate molecular weightsof 65 kilodaltons (Kd), 85-95 Kd, and 200-280 Kd. FIG. 3 represents anautoradiogram of TGF-B cross-linked cell receptors from both thymocytesand the NRK cell line. Approximately 1×10⁷ thymocytes (lanes 1-4) or5×10 NRK cells (lanes 5-12) were used. Lanes 2 and 6 received 20 nMnon-labeled TGF-Bl and lane 10 received 20 nM unlabeled TGF-B2 to showthe specificity of binding. Lanes 3, 7 and 11 received 1D11.16 MAb. Theaddition of control mouse IgG (negative control, lanes 4, 8 and 12) didnot affect TGF-B binding to its receptors. Binding of both TGF-B1 andTGF-B2 to the cellular receptors is effectively inhibited by the MAb1D11.16 (lanes 3, 7, and 11). Only the binding to the 65 Kd receptor isnot completely blocked by the antibody. The ability of both the highmolecular weight receptor and the lower molecular weight receptors to beinhibited suggests that the binding epitope on TGF-B for these receptorsis similar. Since the MAb 1D11.16 blocks binding to all receptors, oneis not able to distinguish between the receptors with respect tofunction.

EXAMPLE 7 (Immunoprecipitation of Iodinated TGF-B2)

The ability of the different MAbs to precipitate iodinated TGF-B2 wasassessed TGF-B2 was labeled with ¹²⁵ I!-TGF-B2 by the chloramine Tmethod (Frolik et al, J Biol Chem (1984) 259:10995). 200 ul ofMAb-containing culture supernatant or purified IgG in 0.02MTris-buffered saline (TBS, pH 8.2) containing 1% sodium deoxycholate(Sigma)(DOC) was mixed with 100,000 cpm (specific activity 125 uCi/ul)of ¹²⁵ I!-TGF-B2 for 2 hr at room temperature. To this solution, 50 ulof a 50% suspension of goat anti-mouse IgG-agarose (Zymed) was added inTBS-DOC buffer for an additional 45 min with rocking. The agarose beadswere washed 3X by centrifugation in TBS-DOC. The pelleted agararose wascounted in a gamma counter (Beckman Gamma 5500, Palo Alto, Calif.).Addition of 1% DOC was necessary to limit nonspecific binding of theTGF-B to anti-Ig-agarose.

FIG. 4 shows that the immune complexes are brought down most effectivelyby the antibody 3C7:14. Counts are expressed as percent specific counts(CPM sample minus nonspecific background/total CPM minus non-specificbackground). A fair amount of batch to batch variation in the percentageof iodinated TGF-B2 brought down by all the MAbs was observed. Thissuggests that the chloramine T iodination procedure may convert aconsiderable amount of the TGF-B into an antigenically alteredconformation.

EXAMPLE 8 (Antibody purification)

The MAbs were purified either from culture supernatant or ascites byprotein A-Sepharose chromatography (Goding, J Immunol Meth (1976) 42:17)(Pharmacia Fine Chemicals, Uppsala, Sweden). The binding of the gamma 1subclass MAbs, 1D11.16 and 2G1.1.12, to protein A was enhanced byaddition of a commercially prepared binding buffer (Bio-Rad, Richmond,Calif.). Antibodies were eluted from the protein A-Sepharose with 0.05Mglycine-HCl plus 0.15M NaCl buffer (pH 2.3), dialyzed overnight versusPBS and stored at -20° C. The gamma 1 subclass antibodies purified fromsupernatants were concentrated and partially purified by ammoniumsulfate precipitation (50% saturated) prior to protein A-chromatography.

EXAMPLE 9 (Competitive RIA)

This solid phase RIA follows the procedure of Mariani et al, J ImmunolMeth (1984) 71:43. Briefly, wells of a 96 well microtiter plate(Microtest III, Becton Dickenson, Oxnard, Calif.) were coated withpurified MAbs at 100 ug/well overnight in 0.01 M carbonate coatingbuffer (pH 9.6). The wells were washed with PT and blocked with PTcontaining 1% (w/v) gelatin for 60 min. The plates were then washed withPT and incubated with 2 fold dilutions of a solution containingnon-labeled TGF-B2 (starting at 20 ug/mL) in PT plus 1% gelatin. To eachwell was added a constant amount of ¹²⁵ I!-TGF-B2 (100,000 cpm; specificactivity 100-150 uCi/ug). After 2-5 hr at room temperature, the platewas thoroughly washed. Individual wells were cut from the plate andcounted in a gamma counter. Calculation of affinity constants wasperformed using a nonlinear regression analysis program (Antoni andMariani, J Immunol Meth (1985) 83:61). The affinity constants, providedin Table 1, ranged from 1.6×10⁷ L/mol to 3.4×10⁸ L/mol.

EXAMPLE 10 (Acute Liver Injury)

A hepatotoxin, D-galactosamine, causes reproducible mortality, liverfibrosis, and maximal TGF-B gene expression approximately 48 hours afteradministration. A rat model utilizing this hepatotoxin is useful forevaluation of TGF-B antibody therapeutic effects on acute liverfibrosis.

Twenty Sprague-Dawley rats were administered 1.6 g/Kg D-galactosamineintraperitoneally. Ten of the rats also received 2 mg/Kg TGF-B antibody(1D11.16) 2 hours prior to hepatotoxin administration, and at 24, 48,and 72 hours post administration. Two rats from each test group weresacrificed at 48 hours to evaluate antibody efficacy at peak TGF-B geneexpression.

Histological examination revealed that anti-TGF-B treated animalsexhibited reduced liver pathology. Northern blot evaluation of tissuesfrom specimens treated with TGF-B antibody showed significantlydecreased levels of collagen mRNA, and almost normal levels of serumalbumin, in contrast to non-treated controls.

Modifications of the above-described modes for carrying out theinvention that are obvious to those of skill in the fields of hybridomatechnology, immunology, and related fields, are intended to be withinthe scope of the following claims.

We claim:
 1. A method for treating or reducing the likelihood ofdeveloping acute or chronic fibrosis, said method comprisingadministering a therapeutically effective amount of a monoclonalantibody that neutralizes transforming growth factor-β1 and transforminggrowth factor-β2.
 2. The method of claim 1 wherein said monoclonalantibody having a binding affinity for transforming growth factor β1 andtransforming growth factor-β2 of at least 10⁷ M⁻¹.
 3. The method ofclaim 1 wherein said monoclonal antibody is a murine antibody.
 4. Themethod of claim 1 wherein said monoclonal antibody is obtained fromhybridoma 1D11.16 having ATCC Accession No. HB9849.
 5. The method ofclaim 1 wherein said monoclonal antibody is an antigen-binding fragmentof a monoclonal antibody.
 6. The method of claim 1 wherein saidmonoclonal antibody is a chimeric monoclonal antibody, said chimericmonoclonal antibody comprising an antigen binding portion and aremainder portion, said antigen binding portion obtained from amonoclonal antibody that neutralizes transforming growth factor-β1 andtransforming growth factor-β2 and said remainder portion obtained fromhuman antibodies.
 7. The method of claim 6 wherein said chimericmonoclonal antibody comprises the antigen binding portion obtained fromthe monoclonal antibody obtained from hybridoma 1D11.16 having A.T.C.C.Accession No. HB 9849.